Both HMGB1 and Ku proteins were previously thought to function as nuclear factors that bind DNA and enhance transcription. Genetic evidence reveals that loss of Ku proteins largely abrogates DNA double-stranded breaks (DSBs) repair and results in a severe defect in the development of B and T cells, whereas loss of HMGB1 leads to a phenotype of postnatal lethality with an unidentified reason. In addition, recent discoveries demonstrate that Ku70 is a mammalian receptor for Rickettsia conorii, and HMGB1 acts as a crucial cytokine that mediates the response to infection, injury and inflammation, thereby establishing a link between HMGB1 and Ku proteins as well as innate immunity. However, the biological functions of HMGB1 and Ku proteins in activation of the TLR pathway have not been explored. Moreover, recent findings have suggested that autoantibody/autoantigen/DNA complexes from the serum of SLE patients induce the pro-inflammatory and type I IFN response, which is involved in the immunopathogenesis of SLE in TLR9-dependent and independent manners. Intriguingly, both HMGB1 and Ku proteins are autoantigens of SLE, and autoantibodies against both of them are present in the serum of some SLE patients. Could HMGB1 and Ku proteins play a role in the antibody/antigen/DNA complex-mediated inflammatory and type I IFN response in SLE? Our results indicate that HMGB1 and Ku proteins are important for the pro-inflammatory cytokine and type I IFN response to immunostimulatory single-stranded DNA (ISS-DNA). However, details of molecular mechanisms are still missing. Thus, we have formulated three specific aims to elucidate the mechanism by which HMGB1 and Ku proteins are required for the innate immune response to ISS-DNA. We believe that our study will provide a better understanding of how HMGB1 and Ku autoantigens are involved in the cytokine response to DNAs, and how TLR9 is activated by ISS-DNA. Moreover, our study will reveal a link between the TLR9- dependent and -independent pathways. Finally, our study will provide new insight information on design of better adjuvants for vaccine against allergy, asthma, cancer and infectious diseases, while providing better inhibitors for treatment of SLE.